Serum and Vitreous miRNA Profiling

Total RNA was extracted from serum and vitreous samples using miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. Gene tip miRNA was extracted from the serum and vitreous samples using 3D-Gene^®RNA extraction reagent from a liquid sample kit (Toray Industries, Inc., Kamakura, Kanagawa, Japan) and concentrated. Fluorescent labeling of RNA was performed using 3D-Gene^® miRNA Labeling kit. Labeled RNA was hybridized to a 3D-Gene^® Human miRNA Oligo Chip (Toray Industries, Inc.) designed to detect 2565 mature human miRNA sequences registered in miRBase release 21 (http://www.mirbase.org/). The chip was scanned using a 3D-Gene^® Scanner, miRNAs with signals higher than the background signal were selected (positive call), and only miRNAs with positive call were used in subsequent analyses. The miRNA signal values were standardized by global normalization (log conversion of data and median alignment) [22 (link)].